Crystalline forms of a prolyl hydroxylase inhibitor

ABSTRACT

The present disclosure relates to crystalline solid forms of [(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-acetic acid, the process of preparing the forms, and pharmaceutical compositions and methods of use thereof.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 U.S.C. §119(e) to U.S.Application No. 61/672,191, filed Jul. 16, 2012, Application No.61/768,297, filed Feb. 22, 2013, and Application No. 61/832,566, filedJun. 7, 2013, all of which are incorporated herein by reference.

FIELD

The present disclosure relates to crystalline solid forms of[(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid, the process of preparing the forms, and pharmaceuticalcompositions and methods of use thereof.

STATE OF THE ART

A compound can exist in one or more crystalline forms. Crystalline formsof a drug substance can have different physical properties, includingmelting point, solubility, dissolution rate, optical and mechanicalproperties, vapor pressure, hygroscopicity, particle shape, density, andflowability. These properties can have a direct effect on the ability toprocess and/or manufacture a compound as a drug product. Crystallineforms can also exhibit different stabilities and bioavailability. Themost stable crystalline form of a drug product is often chosen duringdrug development based on the minimal potential for conversion toanother crystalline form and on its greater chemical stability. Toensure the quality, safety, and efficacy of a drug product, it isimportant to choose a crystalline form that is stable, is manufacturedreproducibly, and has favorable physicochemical properties.

[(4-Hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid (hereinafter, Compound A) is a potent inhibitor of hypoxiainducible factor (HIF) prolyl hydroxylase, as described in U.S. Pat. No.7,323,475. HIF prolyl hydroxylase inhibitors are useful for increasingthe stability and/or activity of HIF, and useful for, inter alia,treating and preventing disorders associated with HIF, including anemia,ischemia, and hypoxia.

SUMMARY

The present disclosure fulfills these needs and others by providingcrystalline forms of Compound A, salts, and solvates. The presentdisclosure also provides an amorphous form of Compound A. The presentdisclosure also provides pharmaceutical compositions comprisingamorphous or one or more crystalline forms of Compound A. The disclosurealso provides processes for making the amorphous and crystalline solidforms and methods for using them to treat, and prevent HIF-associateddisorders including conditions involving anemia, ischemia, and hypoxia.

Thus, one embodiment provided is crystalline[(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid (Compound A Form A) characterized by an X-ray powder diffractogramcomprising the following peaks: 8.5, 16.2, and 27.4°2θ±0.2°2θ.

Another embodiment provided is crystalline[(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid hemihydrate (Compound A Form B) characterized by an X-ray powderdiffractogram comprising the following peaks: 4.2, 8.3, and16.6°2θ±0.2°2θ.

Yet another embodiment provided is crystalline[(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid hexafluoropropan-2-ol solvate (Compound A Form C) characterized byan X-ray powder diffractogram comprising the following peaks: 4.5, 13.7,and 16.4°2θ±0.2°2θ.

Yet another embodiment provided is crystalline[(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid DMSO:water solvate (Compound A Form D) characterized by an X-raypowder diffractogram comprising the following peaks: 8.4, 8.5, and16.8°2θ±0.2°2θ.

Yet another embodiment provided is crystalline[(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid sodium salt (Compound A sodium salt) characterized by an X-raypowder diffractogram comprising the following peaks: 5.3, 16.0, and21.6°2θ±0.2°2θ.

Yet another embodiment provided is crystalline[(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid L-arginine salt (Compound A L-arginine salt) characterized by anX-ray powder diffractogram comprising the following peaks: 20.8, 21.8,and 25.4°2θ±0.2°2θ.

Yet another embodiment provided is crystalline[(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid L-lysine salt (Compound A L-lysine salt) characterized by an X-raypowder diffractogram comprising the following peaks: 19.8, 20.7, and21.2°2θ±0.2°2θ.

Yet another embodiment provided is crystalline[(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid ethanolamine salt (Compound A ethanolamine salt) characterized byan X-ray powder diffractogram comprising the following peaks: 21.8,22.7, and 27.1°2θ±0.2°2θ.

Yet another embodiment provided is crystalline[(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid diethanolamine salt (Compound A diethanolamine salt) characterizedby an X-ray powder diffractogram comprising the following peaks: 16.9,23.7, and 25.0°2θ±0.2°2θ.

Yet another embodiment provided is crystalline[(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid tromethamine salt (Compound A tromethamine salt) characterized byan X-ray powder diffractogram comprising the following peaks: 10.1,14.2, and 21.1°2θ±0.2°2θ.

Yet another embodiment provided is amorphous[(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid (amorphous Compound A).

Yet another embodiment provided is substantially amorphous[(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid potassium salt (Compound A potassium salt).

Still another embodiment provided is directed to a pharmaceuticalcomposition comprising a crystalline or amorphous form of Compound A, ora salt thereof, and a pharmaceutically acceptable excipient.

Additionally, the disclosure provides in one embodiment a method fortreating, pretreating, or delaying onset or progression of a conditionmediated at least in part by hypoxia inducible factor (HIF). The methodcomprises administering to a patient in need thereof a therapeuticallyeffective amount of a compound selected from the group consisting of:Compound A Form A, Compound A Form B, Compound A Form C, Compound A FormD, Compound A sodium salt, Compound A L-arginine salt, Compound AL-lysine salt, Compound A ethanolamine salt, Compound A diethanolaminesalt, Compound A tromethamine salt, amorphous Compound A, and Compound Apotassium salt, as described generally above.

Also provided is a method for treating, pretreating, or delaying onsetor progression of a condition mediated at least in part byerythropoietin (EPO), comprising administering to a patient in needthereof, a therapeutically effective amount of a compound selected fromthe group consisting of: Compound A Form A, Compound A Form B, CompoundA Form C, Compound A Form D, Compound A sodium salt, Compound AL-arginine salt, Compound A L-lysine salt, Compound A ethanolamine salt,Compound A diethanolamine salt, Compound A tromethamine salt, amorphousCompound A, and Compound A potassium salt, as described generally above.

Also provided is a method for treating, pretreating, or delaying onsetor progression of anemia, comprising administering to a patient in needthereof, a therapeutically effective amount of a compound selected fromthe group consisting of: Compound A Form A, Compound A Form B, CompoundA Form C, Compound A Form D, Compound A sodium salt, Compound AL-arginine salt, Compound A L-lysine salt, Compound A ethanolamine salt,Compound A diethanolamine salt, Compound A tromethamine salt, amorphousCompound A, and Compound A potassium salt, as described generally above.

Also provided is a method of inhibiting the activity of a HIFhydroxylase enzyme, the method comprising bringing into contact the HIFhydroxylase enzyme and a therapeutically effective amount of a compoundselected from the group consisting of: Compound A Form A, Compound AForm B, Compound A Form C, Compound A Form D, Compound A sodium salt,Compound A L-arginine salt, Compound A L-lysine salt, Compound Aethanolamine salt, Compound A diethanolamine salt, Compound Atromethamine salt, amorphous Compound A, and Compound A potassium salt,as described generally above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an X-ray powder diffraction pattern of Compound A Form A.

FIG. 2 is a differential scanning calorimetry (DSC) curve of Compound AForm A.

FIG. 3 is an X-ray powder diffraction pattern of Compound A Form B(bottom) plotted with an X-ray powder diffraction pattern of Compound AForm A (top).

FIG. 4 is a thermogravimetric analysis (TGA) (top) and a differentialscanning calorimetry (DSC) curve (bottom) of Compound A Form B.

FIG. 5 is an X-ray powder diffraction pattern of Compound A Form C(bottom) plotted with an X-ray powder diffraction pattern of Compound AForm A (top).

FIG. 6 is a differential scanning calorimetry (DSC) curve (top) and athermogravimetric analysis (TGA) (bottom) of Compound A Form C.

FIG. 7 is an X-ray powder diffraction pattern of Compound A Form D.

FIG. 8 is a thermogravimetric analysis (TGA) (top) and a differentialscanning calorimetry (DSC) curve (bottom) of Compound A Form D.

FIG. 9 is an X-ray powder diffraction pattern of Compound A sodium saltas isolated (bottom) and at 40° C./75% RH (top).

FIG. 10 is a thermogravimetric analysis (TGA) (top) and a differentialscanning calorimetry (DSC) curve (bottom) of Compound A sodium salt.

FIG. 11 is an X-ray powder diffraction pattern of Compound A L-argininesalt as isolated (bottom) and at 40° C./75% RH (top).

FIG. 12 is a thermogravimetric analysis (TGA) (top) and a differentialscanning calorimetry (DSC) curve (bottom) of Compound A L-arginine salt.

FIG. 13 is an X-ray powder diffraction pattern of Compound A L-lysinesalt as isolated (bottom) and at 40° C./75% RH (top).

FIG. 14 is a thermogravimetric analysis (TGA) (top) and a differentialscanning calorimetry (DSC) curve (bottom) of Compound A L-lysine salt.

FIG. 15 is an X-ray powder diffraction pattern of Compound A Form A(bottom), Compound A ethanolamine salt pattern 1 as isolated (second tobottom), Compound A ethanolamine salt pattern 3 at 40° C./75% RH(middle), Compound A ethanolamine salt pattern 2 as isolated (second totop), and Compound A ethanolamine salt pattern 2 at 40° C./75% RH (top).

FIG. 16 is a thermogravimetric analysis (TGA) (top) and a differentialscanning calorimetry (DSC) curve (bottom) of Compound A ethanolaminesalt.

FIG. 17 is an X-ray powder diffraction pattern of Compound A Form A(bottom), Compound A diethanolamine salt pattern 1 from acetone (secondto bottom), Compound A diethanolamine salt pattern 1 from THF (second totop), and Compound A diethanolamine salt at 40° C./75% RH (pattern 2,top).

FIG. 18 is a thermogravimetric analysis (TGA) (top) and a differentialscanning calorimetry (DSC) curve (bottom) of Compound A diethanolaminesalt.

FIG. 19 is an X-ray powder diffraction pattern of Compound A Form A(bottom), and Compound A tromethamine salt as isolated (middle) and at40° C./75% RH (top).

FIG. 20 is a thermogravimetric analysis (TGA) (top) and a differentialscanning calorimetry (DSC) curve (bottom) of Compound A tromethaminesalt.

FIG. 21 is an X-ray powder diffraction pattern of Compound A potassiumsalt as isolated (bottom) and at 40° C./75% RH (top).

FIG. 22 is a thermogravimetric analysis (TGA) (top) and a differentialscanning calorimetry (DSC) curve (bottom) of Compound A potassium salt.

FIG. 23 is an X-ray powder diffraction pattern of amorphous Compound A.

FIG. 24 is the thermogravimetric analysis (TGA) of Compound A Form A.

FIG. 25 is an X-ray powder diffraction pattern of Compound A Form A(bottom), and Compound A hydrochloric acid salt as isolated (middle) andat 40° C./75% RH (top).

FIG. 26 is a thermogravimetric analysis (TGA) (top) and a differentialscanning calorimetry (DSC) curve (bottom) of Compound A hydrochloricacid salt.

FIG. 27 is an X-ray powder diffraction pattern of Compound A Form A(bottom), and Compound A sulfuric acid salt as isolated (middle) and at40° C./75% RH (top).

FIG. 28 is a thermogravimetric analysis (TGA) (top) and a differentialscanning calorimetry (DSC) curve (bottom) of Compound A sulfuric acidsalt.

FIG. 29 is an X-ray powder diffraction pattern of Compound A Form A(bottom), Compound A methanesulfonic acid salt pattern 1 as isolated(second to bottom) and at 40° C./75% RH (middle), and Compound Amethanesulfonic acid salt pattern 2 as isolated (second to top) and at40° C./75% RH (top).

FIG. 30 is a thermogravimetric analysis (TGA) (top) and a differentialscanning calorimetry (DSC) curve (bottom) of Compound A methanesulfonicacid salt.

FIG. 31 is an X-ray powder diffraction pattern of Compound A Form A(bottom), Compound A bis triethylamine salt as isolated (middle) andCompound A bis triethylamine salt at 40° C./75% RH (top).

FIG. 32 is a thermogravimetric analysis (TGA) (top) and a differentialscanning calorimetry (DSC) curve (bottom) of Compound A bistriethylamine salt.

FIG. 33 is an X-ray powder diffraction pattern of Compound A Form A(bottom), and Compound A hemi calcium salt (second crop) at 40° C./75%RH (top).

FIG. 34 is a thermogravimetric analysis (TGA) (top) and a differentialscanning calorimetry (DSC) curve (bottom) of Compound A hemi calciumsalt.

FIG. 35 is an X-ray powder diffraction pattern of Compound A Form A(bottom), and Compound A hemi magnesium salt (second crop) at 40° C./75%RH (top).

FIG. 36 is a differential scanning calorimetry (DSC) curve of Compound Ahemi magnesium salt.

FIG. 37 is the molecular configuration of Compound A Form A.

DETAILED DESCRIPTION

The compound[(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid (Compound A) is a potent inhibitor of hypoxia inducible factor(HIF) prolyl hydroxylase and has the following formula:

The present disclosure provides crystalline forms of Compound A, saltsof Compound A, and solvates of Compound A. The present disclosure alsoprovides an amorphous form of Compound A. The present disclosure alsoprovides pharmaceutical compositions comprising amorphous or crystallineforms of Compound A. The disclosure also provides processes for makingthe amorphous and crystalline solid forms and methods for using them totreat, and prevent HIF-associated disorders including conditionsinvolving anemia, ischemia, and hypoxia.

Prior to discussing in further detail, the following terms will bedefined.

1. DEFINITIONS

As used herein, the following terms have the following meanings.

The singular forms “a,” “an,” and “the” and the like include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a compound” includes both a single compound and aplurality of different compounds.

The term “about” when used before a numerical designation, e.g.,temperature, time, amount, and concentration, including a range,indicates approximations which may vary by ±10%, ±5% or ±1%.

The term “solvate” refers to a complex formed by the combining ofCompound A and a solvent.

The terms “substantially amorphous” and “mostly amorphous” refer toamorphous Compound A where a small amount of crystalline Compound A maybe present. In some embodiments, the amount of crystalline Compound A isless than about 10%, or less than about 5%, or less than about 2%, orless than about 1%, or less than about 0.2%, or less than about 0.1%.

“Administration” refers to introducing an agent into a patient. Atherapeutic amount can be administered, which can be determined by thetreating physician or the like. An oral route of administration ispreferred for the crystalline forms of Compound A described herein. Therelated terms and phrases “administering” and “administration of”, whenused in connection with a compound or pharmaceutical composition (andgrammatical equivalents) refer both to direct administration, which maybe administration to a patient by a medical professional or byself-administration by the patient, and/or to indirect administration,which may be the act of prescribing a drug. For example, a physician whoinstructs a patient to self-administer a drug and/or provides a patientwith a prescription for a drug is administering the drug to the patient.In any event, administration entails delivery of the drug to thepatient.

“Excipient” as used herein means an inert or inactive substance used inthe production of pharmaceutical products, including without limitationany substance used as a binder, disintegrant, coating,compression/encapsulation aid, cream or lotion, lubricant, parenteral,sweetener or flavoring, suspending/gelling agent, or wet granulationagent. Binders include, e.g., carbopol, povidone, xanthan gum, etc.;coatings include, e.g., cellulose acetate phthalate, ethylcellulose,gellan gum, maltodextrin, etc.; compression/encapsulation aids include,e.g., calcium carbonate, dextrose, fructose, honey, lactose (anhydrateor monohydrate; optionally in combination with aspartame, cellulose, ormicrocrystalline cellulose), starch, sucrose, etc.; disintegrantsinclude, e.g., croscarmellose sodium, gellan gum, sodium starchglycolate, etc.; creams and lotions include, e.g., maltodextrin,carrageenans, etc.; lubricants include, e.g., magnesium stearate,stearic acid, sodium stearyl fumarate, etc.; materials for chewabletablets include, e.g., dextrose, fructose dc, lactose (monohydrate,optionally in combination with aspartame or cellulose), etc.;parenterals include, e.g., mannitol, povidone, etc.; plasticizersinclude, e.g., dibutyl sebacate, polyvinylacetate phthalate, etc.;suspending/gelling agents include, e.g., carrageenan, sodium starchglycolate, xanthan gum, etc.; sweeteners include, e.g., aspartame,dextrose, fructose, sorbitol, sucrose, etc.; and wet granulation agentsinclude, e.g., calcium carbonate, maltodextrin, microcrystallinecellulose, etc.

“Therapeutically effective amount” or “therapeutic amount” refers to anamount of a drug or an agent that when administered to a patientsuffering from a condition, will have the intended therapeutic effect,e.g., alleviation, amelioration, palliation or elimination of one ormore manifestations of the condition in the patient. The therapeuticallyeffective amount will vary depending upon the subject and the conditionbeing treated, the weight and age of the subject, the severity of thecondition, the particular composition or excipient chosen, the dosingregimen to be followed, timing of administration, the manner ofadministration and the like, all of which can be determined readily byone of ordinary skill in the art. The full therapeutic effect does notnecessarily occur by administration of one dose, and may occur onlyafter administration of a series of doses. Thus, a therapeuticallyeffective amount may be administered in one or more administrations. Forexample, and without limitation, a therapeutically effective amount ofan agent, in the context of treating anemia, refers to an amount of theagent that alleviates, ameliorates, palliates, or eliminates one or moresymptoms of anemia in the patient.

“Treatment”, “treating”, and “treat” are defined as acting upon adisease, disorder, or condition with an agent to reduce or amelioratethe harmful or any other undesired effects of the disease, disorder, orcondition and/or its symptoms. Treatment, as used herein, covers thetreatment of a human patient, and includes: (a) reducing the risk ofoccurrence of the condition in a patient determined to be predisposed tothe disease but not yet diagnosed as having the condition, (b) impedingthe development of the condition, and/or (c) relieving the condition,i.e., causing regression of the condition and/or relieving one or moresymptoms of the condition.

An “XRPD pattern” is an x-y graph with diffraction angle (i.e., °2θ) onthe x-axis and intensity on the y-axis. The peaks within this patternmay be used to characterize a crystalline solid form. As with any datameasurement, there is variability in XRPD data. The data are oftenrepresented solely by the diffraction angle of the peaks rather thanincluding the intensity of the peaks because peak intensity can beparticularly sensitive to sample preparation (for example, particlesize, moisture content, solvent content, and preferred orientationeffects influence the sensitivity), so samples of the same materialprepared under different conditions may yield slightly differentpatterns; this variability is usually greater than the variability indiffraction angles. Diffraction angle variability may also be sensitiveto sample preparation. Other sources of variability come from instrumentparameters and processing of the raw X-ray data: different X-rayinstruments operate using different parameters and these may lead toslightly different XRPD patterns from the same solid form, and similarlydifferent software packages process X-ray data differently and this alsoleads to variability. These and other sources of variability are knownto those of ordinary skill in the pharmaceutical arts. Due to suchsources of variability, it is usual to assign a variability of ±0.2°2θto diffraction angles in XRPD patterns.

2. SOLID FORMS OF COMPOUND A

As described generally above, the present disclosure provides solidforms of[(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid (Compound A).

Compound A Form A is characterized by its X-ray powder diffractogramthat comprises peaks at 8.5, 16.2, and 27.4°2θ±0.2°2θ. The diffractogramcomprises additional peaks at 12.8, 21.6, and 22.9°2θ±0.2°2θ. Form Aalso is characterized by its full X-ray powder diffractogram assubstantially shown in FIG. 1.

In some embodiments, Form A is characterized by its differentialscanning calorimetry (DSC) curve that comprises an endotherm at about223° C. Form A also is characterized by its full DSC curve assubstantially as shown in FIG. 2.

Compound A Form B is characterized by its X-ray powder diffractogramthat comprises peaks at 4.2, 8.3, and 16.6°2θ±0.2°2θ. The diffractogramcomprises additional peaks at 12.5, 14.1, and 17.4°2θ±0.2°2θ. Form Balso is characterized by its full X-ray powder diffractogram assubstantially shown in FIG. 3.

In some embodiments, Form B is characterized by its differentialscanning calorimetry (DSC) curve that comprises an endotherm at about222° C. Form B also is characterized by its full DSC curve assubstantially as shown in FIG. 4.

Compound A Form C is characterized by its X-ray powder diffractogramthat comprises peaks at 4.5, 13.7, and 16.4°2θ±0.2°2θ. The diffractogramcomprises additional peaks at 15.4, 15.5, and 20.6°2θ±0.2°2θ. Form Calso is characterized by its full X-ray powder diffractogram assubstantially shown in FIG. 5.

In some embodiments, Form C is characterized by its differentialscanning calorimetry (DSC) curve that comprises an endotherm at about222° C. Form C also is characterized by its full DSC curve assubstantially as shown in FIG. 6.

Compound A Form D is characterized by its X-ray powder diffractogramthat comprises peaks at 8.4, 8.5, and 16.8°2θ±0.2°2θ. The diffractogramcomprises additional peaks at 4.2, 12.6, and 28.4°2θ±0.2°2θ. Form D alsois characterized by its full X-ray powder diffractogram as substantiallyshown in FIG. 7.

In some embodiments, Form D is characterized by its differentialscanning calorimetry (DSC) curve that comprises an endotherm at about222° C. Form D also is characterized by its full DSC curve assubstantially as shown in FIG. 8.

Compound A sodium salt is characterized by its X-ray powderdiffractogram that comprises peaks at 5.3, 16.0, and 21.6°2θ±0.2°2θ. Thediffractogram comprises additional peaks at 18.7, 19.2, and24.0°2θ±0.2°2θ. Compound A sodium salt also is characterized by its fullX-ray powder diffractogram as substantially shown in FIG. 9.

In some embodiments, Compound A sodium salt is characterized by itsdifferential scanning calorimetry (DSC) curve that comprises anendotherm at about 314° C. Compound A sodium salt also is characterizedby its full DSC curve as substantially as shown in FIG. 10.

Compound A L-arginine salt is characterized by its X-ray powderdiffractogram that comprises peaks at 20.8, 21.8, and 25.4°2θ±0.2°2θ.The diffractogram comprises additional peaks at 22.7, 23.4, and26.4°2θ±0.2°2θ. Compound A L-arginine salt also is characterized by itsfull X-ray powder diffractogram as substantially shown in FIG. 11.

In some embodiments, Compound A L-arginine salt is characterized by itsdifferential scanning calorimetry (DSC) curve that comprises anendotherm at about 210° C. Compound A L-arginine salt also ischaracterized by its full DSC curve as substantially as shown in FIG.12.

Compound A L-lysine salt is characterized by its X-ray powderdiffractogram that comprises peaks at 19.8, 20.7, and 21.2°2θ±0.2°2θ.The diffractogram comprises additional peaks at 10.2, 16.9, and18.4°2θ±0.2°2θ. Compound A L-lysine salt also is characterized by itsfull X-ray powder diffractogram as substantially shown in FIG. 13.

In some embodiments, Compound A L-lysine salt is characterized by itsdifferential scanning calorimetry (DSC) curve that comprises anendotherm at about 237° C. Compound A L-lysine salt also ischaracterized by its full DSC curve as substantially as shown in FIG.14.

Compound A ethanolamine salt is characterized by its X-ray powderdiffractogram that comprises peaks at 21.8, 22.7, and 27.1°2θ±0.2°2θ.The diffractogram comprises additional peaks at 21.1, 26.2, and26.6°2θ±0.2°2θ. Compound A ethanolamine salt also is characterized byits full X-ray powder diffractogram as substantially shown in FIG. 15.

In some embodiments, Compound A ethanolamine salt is characterized byits differential scanning calorimetry (DSC) curve that comprises anendotherm at about 171° C. Compound A ethanolamine salt also ischaracterized by its full DSC curve as substantially as shown in FIG.16.

Compound A diethanolamine salt is characterized by its X-ray powderdiffractogram that comprises peaks at 16.9, 23.7, and 25.0°2θ±0.2°2θ.The diffractogram comprises additional peaks at 19.6, 22.6, and26.0°2θ±0.2°2θ. Compound A diethanolamine salt also is characterized byits full X-ray powder diffractogram as substantially shown in FIG. 17.

In some embodiments, Compound A diethanolamine salt is characterized byits differential scanning calorimetry (DSC) curve that comprises anendotherm at about 150° C. Compound A diethanolamine salt also ischaracterized by its full DSC curve as substantially as shown in FIG.18.

Compound A tromethamine salt is characterized by its X-ray powderdiffractogram that comprises peaks at 10.1, 14.2, and 21.1°2θ±0.2°2θ.The diffractogram comprises additional peaks at 20.1, 25.7, and28.4°2θ±0.2°2θ. Compound A tromethamine salt also is characterized byits full X-ray powder diffractogram as substantially shown in FIG. 19.

In some embodiments, Compound A tromethamine salt is characterized byits differential scanning calorimetry (DSC) curve that comprises anendotherm at about 176° C. Compound A tromethamine salt also ischaracterized by its full DSC curve as substantially as shown in FIG.20.

Also provided is amorphous[(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid (amorphous Compound A) and substantially amorphous[(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid potassium salt (Compound A potassium salt). The substantiallyamorphous Compound A potassium salt has been characterized by adifferential scanning calorimetry (DSC) curve that comprises anendotherm at about 291° C. (FIG. 22).

As described in the Examples below, Form A is the most stablecrystalline form among Form B, C, and D of Compound A.

3. PHARMACEUTICAL COMPOSITIONS, FORMULATIONS AND ROUTES OFADMINISTRATION

In one aspect, the present disclosure is directed to a pharmaceuticalcomposition comprising one or more crystalline forms of[(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid (Compound A) having the following structure:

or a salt thereof, and at least one pharmaceutically acceptableexcipient.

In one embodiment, the pharmaceutical composition comprises a compoundselected from the group consisting of: Compound A Form A, Compound AForm B, Compound A Form C, Compound A Form D, Compound A sodium salt,Compound A L-arginine salt, Compound A L-lysine salt, Compound Aethanolamine salt, Compound A diethanolamine salt, Compound Atromethamine salt, amorphous Compound A, and Compound A potassium salt,as described generally above, and at least one pharmaceuticallyacceptable excipient.

In one embodiment, the pharmaceutical composition comprises Compound Ain Form A. In one embodiment, the pharmaceutical composition comprisesCompound A wherein at least about 85% of Compound A is in Form A. In oneembodiment, the pharmaceutical composition comprises Compound A whereinat least about 90% of Compound A is in Form A. In one embodiment, thepharmaceutical composition comprises Compound A wherein at least about95% of Compound A is in Form A. In one embodiment, the pharmaceuticalcomposition comprises Compound A wherein at least about 99% of CompoundA is in Form A. In one embodiment, the pharmaceutical compositioncomprises Compound A wherein at least about 99.5% of Compound A is inForm A. In one embodiment, the pharmaceutical composition comprisesCompound A wherein at least about 99.9% of Compound A is in Form A. Inone embodiment, the pharmaceutical composition comprises Compound Awherein at least about 99.99% of Compound A is in Form A.

In one embodiment, the pharmaceutical composition further comprises anadditional therapeutic agent selected from the group consisting ofvitamin B12, folic acid, ferrous sulfate, recombinant humanerythropoietin, and an erythropoiesis stimulating agent (ESA). Inanother embodiment, the pharmaceutical composition is formulated fororal delivery. In another embodiment, the pharmaceutical composition isformulated as a tablet or a capsule.

The crystalline forms of the present disclosure can be delivereddirectly or in pharmaceutical compositions along with suitableexcipients, as is well known in the art. Various treatments embodiedherein can comprise administration of an effective amount of acrystalline form of the disclosure to a subject in need, e.g., a subjecthaving or at risk for anemia due to, e.g., chronic renal failure,diabetes, cancer, AIDS, radiation therapy, chemotherapy, kidneydialysis, or surgery. In one embodiment, the subject is a mammaliansubject, and in one embodiment, the subject is a human subject.

An effective amount of a crystalline form can readily be determined byroutine experimentation, as can the most effective and convenient routeof administration and the most appropriate formulation. In oneembodiment, the dosage may be from 0.05 mg/kg to about 700 mg/kg perday. Typically, the dosage may be from about 0.1 mg/kg to about 500mg/kg; from about 0.5 mg/kg to about 250 mg/kg; from about 1 mg/kg toabout 100 mg/kg; from about 1 mg/kg to about 10 mg/kg; from about 1mg/kg to about 5 mg/kg; or from about 1 mg/kg to about 2 mg/kg. Forexample, the dosage may be about 1.0 mg/kg; about 1.2 mg/kg; about 1.5mg/kg; about 2.0 mg/kg; or about 2.5 mg/kg. Various formulations anddrug delivery systems are available in the art (see, e.g., Gennaro, A.R., ed. (1995) Remington's Pharmaceutical Sciences).

Suitable routes of administration may, for example, include oral,rectal, transmucosal, nasal, or intestinal administration and parenteraldelivery, including intramuscular, subcutaneous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intravenous, intraperitoneal, intranasal, or intraocular injections. Thecrystalline form or composition thereof may be administered in a localrather than a systemic manner. For example, a crystalline form orcomposition thereof can be delivered via injection or in a targeted drugdelivery system, such as a depot or sustained release formulation. Inone embodiment, the route of administration is oral.

The pharmaceutical compositions of the present disclosure may bemanufactured by any of the methods well-known in the art, such as byconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes. Asnoted above, the compositions can include one or more pharmaceuticallyacceptable excipients that facilitate processing of active moleculesinto preparations for pharmaceutical use.

Proper formulation is dependent upon the route of administration chosen.For injection, for example, the composition may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks' solution, Ringer's solution, or physiological saline buffer. Fortransmucosal or nasal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art. In a preferred embodiment of the presentdisclosure, the present crystalline forms are prepared in a formulationintended for oral administration. For oral administration, it can beformulated readily by combining the crystalline forms withpharmaceutically acceptable excipients well known in the art. Suchexcipients enable the crystalline forms of the disclosure to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries, suspensions and the like, for oral ingestion by a subject. Thecrystalline forms may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

Pharmaceutical preparations for oral use can be obtained using solidexcipients, optionally grinding a resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients are, for example,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, microcrystalline cellulose and/orpolyvinylpyrrolidone (PVP or povidone). If desired, disintegratingagents may be added, such as the cross-linked polyvinyl pyrrolidone,agar, croscarmellose sodium or alginic acid or a salt thereof such assodium alginate. Also, wetting agents such as sodium dodecyl sulfate orlubricants such as magnesium stearate may be included.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active doses.

Pharmaceutical preparations for oral administration include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the crystalline forms may be dissolved or suspended insuitable liquids, such as fatty oils, liquid paraffin, or liquidpolyethylene glycols. In addition, stabilizers may be added. Allformulations for oral administration should be in dosages suitable forsuch administration.

In one embodiment, the crystalline forms described herein can beadministered transdermally, such as through a skin patch, or topically.In one aspect, the transdermal or topical formulations can additionallycomprise one or multiple penetration enhancers or other effectors,including agents that enhance migration of the delivered compound.Transdermal or topical administration could be preferred, for example,in situations in which location specific delivery is desired.

For administration by inhalation, the crystalline forms for useaccording to the present disclosure are conveniently delivered in theform of an aerosol spray presentation from pressurized packs or anebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide, or any other suitable gas. Inthe case of a pressurized aerosol, the appropriate dosage unit may bedetermined by providing a valve to deliver a metered amount. Capsulesand cartridges of, for example, gelatin, for use in an inhaler orinsufflator may be formulated. These typically contain a powder mix ofthe crystalline form and a suitable powder base such as lactose orstarch.

Compositions formulated for parenteral administration by injection,e.g., by bolus injection or continuous infusion can be presented in unitdosage form, e.g., in ampoules or in multi-dose containers, with anadded preservative. The compositions may take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Formulations for parenteral administration include aqueoussolutions or other compositions in water-soluble form.

Suspensions of the crystalline forms may also be prepared as appropriateoily injection suspensions. Suitable lipophilic solvents or vehiclesinclude fatty oils such as sesame oil and synthetic fatty acid esters,such as ethyl oleate or triglycerides, or liposomes. Aqueous injectionsuspensions may contain substances that increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents that increase the solubility of the crystallineforms to allow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

As mentioned above, the compositions of the present disclosure may alsobe formulated as a depot preparation. Such long acting formulations maybe administered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thepresent crystalline forms may be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

For any composition used in the various treatments embodied herein, atherapeutically effective dose can be estimated initially using avariety of techniques well known in the art. For example, in a cellculture assay, a dose can be formulated in animal models to achieve acirculating concentration range that includes the IC₅₀ as determined incell culture. Dosage ranges appropriate for human subjects can bedetermined, for example, using data obtained from cell culture assaysand non-human animal studies. In one embodiment, the dosage may be from0.05 mg/kg to about 700 mg/kg administered periodically. The dosage maybe administered once a day, every other day, one, two or three times aweek, or at other appropriate intervals as can be readily determined bycompetent medical practitioners. Typically the dosage is administered 2or 3 times a week. Typically, the dosage may be from about 0.1 mg/kg toabout 500 mg/kg; from about 0.5 mg/kg to about 250 mg/kg; from about 1mg/kg to about 100 mg/kg; from about 1 mg/kg to about 10 mg/kg; fromabout 1 mg/kg to about 5 mg/kg; or from about 1 mg/kg to about 2 mg/kg.For example, the dosage may be about 1.0 mg/kg; about 1.2 mg/kg; about1.5 mg/kg; about 2.0 mg/kg; or about 2.5 mg/kg.

A therapeutically effective dose of a compound refers to that amount ofthe compound that results in amelioration of symptoms or a prolongationof survival in a subject. Toxicity and therapeutic efficacy of suchmolecules can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., by determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratio oftoxic to therapeutic effects is the therapeutic index, which can beexpressed as the ratio LD₅₀/ED₅₀. Compounds that exhibit hightherapeutic indices are preferred.

Dosages preferably fall within a range of circulating concentrationsthat includes the ED₅₀ with little or no toxicity. Dosages may varywithin this range depending upon the dosage form employed and the routeof administration utilized. The exact formulation, route ofadministration, and dosage should be chosen, according to methods knownin the art, in view of the specifics of a subject's condition.

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety that are sufficient to modulate adesired parameter, e.g., endogenous erythropoietin plasma levels, i.e.minimal effective concentration (MEC). The MEC will vary for eachcompound but can be estimated from, for example, in vitro data. Dosagesnecessary to achieve the MEC will depend on individual characteristicsand route of administration. Compounds or compositions thereof should beadministered using a regimen which maintains plasma levels above the MECfor about 10-90% of the duration of treatment, preferably about 30-90%of the duration of treatment, and most preferably between 50-90%. Incases of local administration or selective uptake, the effective localconcentration of the drug may not be related to plasma concentration.Alternatively, modulation of a desired parameter, e.g., stimulation ofendogenous erythropoietin, may be achieved by 1) administering a loadingdose followed by a maintenance dose, 2) administering an induction doseto rapidly achieve the desired parameter, e.g., erythropoietin levels,within a target range, followed by a lower maintenance dose to maintain,e.g., hematocrit, within a desired target range, or 3) repeatedintermittent dosing.

The amount of compound or composition administered will, of course, bedependent on a variety of factors, including the sex, age, and weight ofthe subject being treated, the severity of the affliction, the manner ofadministration, and the judgment of the prescribing physician.

The present compositions may, if desired, be presented in a pack ordispenser device containing one or more unit dosage forms containing theactive ingredient. Such a pack or device may, for example, comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.Compositions comprising a crystalline form of the disclosure formulatedin a compatible pharmaceutical excipient may also be prepared, placed inan appropriate container, and labeled for treatment of an indicatedcondition. Suitable conditions indicated on the label may includetreatment of conditions, disorders, or diseases in which anemia is amajor indication.

4. METHOD OF USE

One aspect of the disclosure provides for use of one or more of acrystalline or amorphous form of[(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid (Compound A), or a composition comprising one or more crystallineor amorphous forms of Compound A or a solvate or salt thereof, for themanufacture of a medicament for use in treating various conditions ordisorders as described herein. It also provides methods of using thecrystalline or amorphous form, or composition or medicament thereof, totreat, pretreat, or delay progression or onset of various conditions ordisorders as described herein. In one embodiment, the crystalline formof Compound A used in the method is Form A. In one embodiment, thecrystalline form of Compound A used in the method is Form B, Form C orForm D. As used in this section “Method of Use” and the correspondingmethod claims, “compound” refers to a crystalline or amorphous form ofCompound A, or a solvate or salt thereof.

In one embodiment, at least about 85% of the compound used in the methodis Compound A Form A. In one embodiment, at least about 90% of thecompound used in the method is Compound A Form A. In one embodiment, atleast about 95% of the compound used in the method is Compound A Form A.In one embodiment, at least about 99% of the compound used in the methodis Compound A Form A. In one embodiment, at least about 99.5% of thecompound used in the method is Compound A Form A. In one embodiment, atleast about 99.9% of the compound used in the method is Compound A FormA. In one embodiment, at least about 99.99% of the compound used in themethod is Compound A Form A.

The medicaments or compositions can be used to modulate the stabilityand/or activity of HIF, and thereby activate HIF-regulated geneexpression. The crystalline or amorphous form, or composition ormedicament thereof, can be used in methods to treat, pretreat, or delayprogression or onset of conditions associated with HIF including, butnot limited to, anemic, ischemic, and hypoxic conditions. In variousembodiments, the crystalline or amorphous form, or composition ormedicament thereof, is administered immediately following a conditionproducing acute ischemia, e.g., myocardial infarction, pulmonaryembolism, intestinal infarction, ischemic stroke, and renalischemic-reperfusion injury. In another embodiment, the crystalline oramorphous form, or composition or medicament thereof, is administered toa patient diagnosed with a condition associated with the development ofchronic ischemia, e.g., cardiac cirrhosis, macular degeneration,pulmonary embolism, acute respiratory failure, neonatal respiratorydistress syndrome, and congestive heart failure. In yet anotherembodiment, the crystalline or amorphous form, or composition ormedicament thereof, is administered immediately after a trauma orinjury. In other embodiments, the crystalline or amorphous form, orcomposition or medicament thereof, can be administered to a subjectbased on predisposing conditions, e.g., hypertension, diabetes,occlusive arterial disease, chronic venous insufficiency, Raynaud'sdisease, chronic skin ulcers, cirrhosis, congestive heart failure, andsystemic sclerosis. In still other embodiments, the crystalline oramorphous form, or composition or medicament thereof, may beadministered to pretreat a subject to decrease or prevent thedevelopment of tissue damage associated with ischemia or hypoxia.

The crystalline or amorphous form, or compositions or medicamentsthereof, can also be used to increase endogenous erythropoietin (EPO).The crystalline or amorphous form, or composition or medicament thereof,can be administered to prevent, pretreat, or treat EPO-associatedconditions, including, e.g., conditions associated with anemia andneurological disorders. Conditions associated with anemia includedisorders such as acute or chronic kidney disease, diabetes, cancer,ulcers, infection with virus, e.g., HIV, bacteria, or parasites;inflammation, etc. Anemic conditions can further include thoseassociated with procedures or treatments including, e.g., radiationtherapy, chemotherapy, dialysis, and surgery. Disorders associated withanemia additionally include abnormal hemoglobin and/or erythrocytes,such as found in disorders such as microcytic anemia, hypochromicanemia, aplastic anemia, etc.

The disclosure is also directed to use of a crystalline or amorphousform, or composition or medicament thereof, to treat, pretreat, or delayonset of a condition associated with a disorder selected from the groupconsisting of anemic disorders; neurological disorders and/or injuriesincluding cases of stroke, trauma, epilepsy, and neurodegenerativedisease; cardiac ischemia including, but not limited to, myocardialinfarction and congestive heart failure; liver ischemia including, butnot limited to, cardiac cirrhosis; renal ischemia including, but notlimited to, acute kidney failure and chronic kidney failure; peripheralvascular disorders, ulcers, burns, and chronic wounds; pulmonaryembolism; and ischemic-reperfusion injury.

The disclosure is also directed to a method of inhibiting the activityof at least one hydroxylase enzyme which modifies the alpha subunit ofhypoxia inducible factor. The HIF hydroxylase enzyme may be a prolylhydroxylase including, but not limited to, the group consisting ofEGLN1, EGLN2, and EGLN3 (also known as PHD2, PHD1 and PHD3,respectively), described by Taylor (2001, Gene 275:125-132), andcharacterized by Aravind and Koonin (2001, Genome Biol 2:RESEARCH0007),Epstein et al. (2001, Cell 107:43-54), and Bruick and McKnight (2001,Science 294:1337-1340). The method comprises contacting the enzyme withan inhibiting effective amount of one or more crystalline or amorphousform of Compound A. In some embodiments, the HIF hydroxylase enzyme isan asparaginyl hydroxylase or a prolyl hydroxylase. In otherembodiments, the HIF hydroxylase enzyme is a factor inhibiting HIF,human EGLN1, EGLN2, or EGLN3.

While this disclosure has been described in conjunction with specificembodiments and examples, it will be apparent to a person of ordinaryskill in the art, having regard to that skill and this disclosure, thatequivalents of the specifically disclosed materials and methods willalso be applicable to this disclosure; and such equivalents are intendedto be included within the following claims.

EXAMPLES

Unless otherwise stated, the following abbreviations used throughout thespecification have the following definitions:

° C. Degree Celsius Ac Acetyl ca. About d Doublet dd Doublet of doubletsDMA Dimethylamine DMEM Eagle's minimal essential medium DMFDimethylformamide DMSO Dimethylsulfoxide DSC Differential scanningcalorimetry EDTA Ethylenediaminetetraacetic acid EtOAc Ethyl Acetate eq.Equivalents FBS Fetal bovine serum g Gram Gly Glycine h Hour HEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HPLC High performanceliquid chromatography IPA Isopropyl alcohol iPrOAc Isopropylacetate JJoules J Coupling constant kg Kilogram kV Kilovolts m Multiplet M MolarM+ Mass peak mA Milliamps Me Methyl MEC Minimal effective concentrationMeCN Acetonitrile MEK Methyl ethyl ketone mg Milligram MHz MegahertzMIBK Methyl iso-butyl ketone min Minute mIU Milliinternational units mLMilliliter mm Millimeter mM Millimolar mol Mole MS Mass spectroscopy NMRNuclear magnetic resonance PBS Phosphate buffer system Ph Phenyl RHRelative humidity rpm Revolutions per minute s Singlet s Second TEATriethylamine TGA Thermogravimetric analysis THF Tetrahydrofuran TsTosyl vol Volume w weight XRPD X-ray powder diffraction δ Chemical shiftμL Microliter μM Micromolar

X-Ray Powder Diffraction (XRPD)

X-Ray Powder Diffraction patterns were collected on a Bruker AXS C2GADDS diffractometer using Cu Kα radiation (40 kV, 40 mA), automated XYZstage, laser video microscope for auto-sample positioning and a HiStar2-dimensional area detector. X-ray optics consists of a single Gobelmultilayer mirror coupled with a pinhole collimator of 0.3 mm. A weeklyperformance check is carried out using a certified standard NIST 1976Corundum (flat plate).

The beam divergence, i.e. the effective size of the X-ray beam on thesample, was approximately 4 mm. A θ-θ continuous scan mode was employedwith a sample—detector distance of 20 cm which gives an effective 2θrange of 3.2°-29.7°. Typically the sample would be exposed to the X-raybeam for 120 seconds. The software used for data collection was GADDSfor WNT 4.1.16 and the data were analyzed and presented using DiffracPlus EVA v11.0.0.2 or v13.0.0.2.

Alternatively, X-Ray Powder Diffraction patterns were collected on aBruker D8 diffractometer using Cu Kα radiation (40 kV, 40 mA), θ-2θgoniometer, and divergence of V4 and receiving slits, a Ge monochromatorand a Lynxeye detector. The instrument is performance checked using acertified Corundum standard (NIST 1976). The software used for datacollection was Diffrac Plus XRD Commander v2.5.0 and the data wereanalyzed and presented using Diffrac Plus EVA v11.0.0.2 or v13.0.0.2.

Samples were run under ambient conditions as flat plate specimens usingpowder as received. The sample was gently packed into a cavity cut intopolished, zero-background (510) silicon wafer. The sample was rotated inits own plane during analysis. The details of the data collection are:

-   -   Angular range: 2 to 42°2θ    -   Step size: 0.05°2θ    -   Collection time: 0.5 s/step    -   Analysis duration: 7 minutes

Differential Scanning Calorimetry (DSC)

DSC was were collected on a TA Instruments Q2000 equipped with a 50position autosampler. The calibration for thermal capacity was carriedout using sapphire and the calibration for energy and temperature wascarried out using certified indium. Typically 0.5-3 mg of each sample,in a pin-holed aluminum pan, was heated at 10° C./min from 25° C. to300° C. A purge of dry nitrogen at 50 ml/min was maintained over thesample. Modulated temperature DSC was carried out using an underlyingheating rate of 2° C./min and temperature modulation parameters of±0.318° C. (amplitude) every 60 seconds (period). The instrument controlsoftware was Advantage for Q Series v2.8.0.392 and Thermal Advantagev4.8.3 and the data were analyzed using Universal Analysis v4.4A.

Alternatively, the DSC data was collected on a Mettler DSC 823e equippedwith a 34 position auto-sampler. The instrument was calibrated forenergy and temperature using certified indium. Typically 0.5-3 mg ofeach sample, in a pin-holed aluminum pan, was heated at 10° C./min from25° C. to 300° C. or 25° C. to 320° C. A nitrogen purge at 50 ml/min wasmaintained over the sample. The instrument control and data analysissoftware was STARe v9.20.

Thermo-Gravimetric Analysis (TGA)

TGA data were collected on a Mettler TGNSDT A 851 e equipped with a 34position autosampler. The instrument was temperature calibrated usingcertified indium. Typically, 1-30 mg of each sample was loaded onto apre-weighed aluminum crucible and was heated at 10° C./min from ambienttemperature to 350° C. A nitrogen purge at 50 ml/min was maintained overthe sample. The instrument control and data analysis software was STARev9.20.

Example 1 Preparation of Compound A Form A Methods

The crystalline[(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid (Compound A Form A) was prepared via the following methods.

Method I

The crystalline Compound A Form A (see Example 1, Method I) was used inthis method. 15 mg of the crystalline material was used with eachsolvent added in increments until a clear solution had been obtained oruntil 50 volumes (750 μL) of solvent had been added. Samples weresonicated for 5 seconds after each solvent addition. When insoluble, theslurries were stirred at 500 rpm cycling between 25° C. and 50° C. (4 hat each temperature) for a period of from 16 hours to six days. Anyresulting solutions were then allowed to evaporate at room temperature.The solids obtained from this experiment were analyzed by XRPD.

Each of the following solvents used in the above described Method Iprovided Form A: acetic acid, acetone, acetophenone, benzonitrile,benzyl alcohol, butyronitrile, chlorobenzene, cyclohexanone,1,2-dichlorobenzene, 1,2-dichloroethane, dimethoxyethane,dimethylacetamide, DMSO, 1,4-dioxane, ethylene glycol, EtOAc, formamide,hexafluorobenzene, hexane, IPA, IPA: 10% water, iPrOAc, MeCN, MEK, MIBK,nitromethane, perfluorohexane, propionitrile, sulfolane, t-butyl methylether, t-butanol, tetraline, THF, and toluene.

Using Method I, hexafluoropropan-2-ol, methanol and ethanol did notprovide Form A.

Method II

The crystalline Compound A Form A (see Example 1, Method VIII) was usedin this method. 15 mg of the crystalline material was used with 30volumes (450 μL) of solvent with the exception of DMSO and DMA where 5volumes were used. Slurries were sonicated for 5 seconds. The slurrieswere stirred at 500 rpm at 5° C. for a period of six days. Any resultingsolutions were then allowed to evaporate at room temperature. The solidsobtained were analyzed by XRPD.

Each of the following solvents used in the above described Method IIprovided Form A: benzonitrile, sulfolane, formamide, tetraline,acetophenone, benzyl alcohol, ethylene glycol, 1,2-dichlorobenzene,chlorobenzene, cyclohexanone, butyronitrile, acetic acid, nitromethane,propionitrile, dimethoxyethane, 1,2-dichloroethane, hexafluorobenzene,t-butanol, hexane, and perfluorohexane.

Using Method II, hexafluoropropan-2-ol did not provide Form A.

Method III

The crystalline Compound A Form A (see Example 1, Method VIII) was usedin this method. Method III is substantially as described in Method II,above, with the exception that the slurries were stirred at 500 rpm at50° C. for a period of six days. The solids obtained were analyzed byXRPD.

Each of the following solvents used in the above described Method IIIprovided Form A: benzonitrile, sulfolane, formamide, tetraline,acetophenone, benzyl alcohol, ethylene glycol, 1,2-dichlorobenzene,chlorobenzene, cyclohexanone, butyronitrile, acetic acid, nitromethane,propionitrile, dimethoxyethane, 1,2-dichloroethane, hexafluorobenzene,t-butanol, hexane, and perfluorohexane.

Using Method III, dimethylacetamide, t-butyl methyl ether, andhexafluoropropan-2-ol did not provide Form A.

Method IV

The crystalline Compound A Form B (see Example 2) was used in thismethod. 15 mg of the crystalline material was used with 30 volumes (450μL) solvent with the exception of DMSO and DMA where 5 volumes was used.Slurries were sonicated for 5 seconds. The slurries were stirred at 500rpm, cycling between 25° C. and 50° C. (4 h at each temperature) for sixdays. Any resulting solutions were then left to evaporate quickly atroom temperature. The solids were analyzed by XRPD.

Each of the following solvents used in the above described Method IVprovided Form A: benzonitrile, sulfolane, formamide, tetraline,acetophenone, benzyl alcohol, ethylene glycol, 1,2-dichlorobenzene,chlorobenzene, cyclohexanone, butyronitrile, acetic acid, t-butyl methylether, nitromethane, propionitrile, dimethoxyethane, 1,2-dichloroethane,hexafluorobenzene, t-butanol, hexane, perfluorohexane, andhexafluoropropan-2-ol.

Method V

The crystalline Compound A Form A (see Example 1, Method VIII) was usedin this method. 20 mg of the crystalline material was dissolved in THF(410 μL) before the addition of 10 volumes (200 μL) of solvent with theexception of DMSO and DMA where 5 volumes was used. The slurries werestirred at 500 rpm cycling between 25° C. and 50° C. (4 h at eachtemperature) for 48 hours. Any solutions which were obtained after theheat/cool cycles were allowed to evaporate at room temperature. Thesolids obtained were analyzed by XRPD.

Each of the following solvents used in the above described Method Vprovided Form A. Benzonitrile, sulfolane, formamide, tetraline,acetophenone, benzyl alcohol, ethylene glycol, DMSO,1,2-dichlorobenzene, chlorobenzene, cyclohexanone, butyronitrile, aceticacid, t-butyl methyl ether, propionitrile, dimethoxyethane,1,2-dichloroethane, hexafluorobenzene, t-butanol, and hexane.

Using Method V, nitromethane, hexafluoropropan-2-ol, and perfluorohexanedid not provide Form A.

Method VI

The crystalline Compound A Form A (30 mg, see Example 1, Method VIII)was dissolved in 10 mL of acetone. This solution was subject to fastsolvent evaporation on a rota-evaporator (40° C., 35-50 Torr). 12.85 mgof the resulting material was used with 10 volumes (128.5 μL) of solventwith the exception of DMSO and DMA where 5 volumes was used. Slurrieswere sonicated for 5 seconds. The slurries were stirred at 500 rpmbetween 25° C. and 50° C. (8 h cycles) for a period of six days. Anyresulting solutions were then allowed to evaporate at room temperature.Solids obtained were analyzed by XRPD.

Each of the following solvents used in the above described Method VIprovided Form A: benzonitrile, sulfolane, formamide, tetraline,acetophenone, benzyl alcohol, ethylene glycol, DMSO,1,2-dichlorobenzene, chlorobenzene, butyronitrile, acetic acid, t-butylmethyl ether, nitromethane, propionitrile, dimethoxyethane,1,2-dichloroethane, hexafluorobenzene, t-butanol, and hexane.

Using Method VI, cyclohexanone, hexafluoropropan-2-ol, andperfluorohexane did not provide Form A.

Method VII

The crystalline Compound A Form A (see Example 1, Method VIII) was usedin this method. 30 mg was suspended in 7 volumes of solvent (10%aqueous). Slurries were sonicated for 5 seconds. The slurries werestirred at 500 rpm cycling between 25° C. and 50° C. (8 h cycles) for aperiod of four days. The solids obtained were analyzed by XRPD.

Each of the following solvents used in the above described Method VIIprovided Form A: acetone, acetonitrile, ethanol, methanol, 2-methyl-THF,and IPA.

Method VIII

An aqueous solution of sodium hydroxide was added slowly to a stirredsuspension of Compound A in water at temperature range (10° C. to 90°C.). A solution of acetic acid in water was then slowly charged attemperature range (10° C. to 90° C.) and the mixture was stirred. Thesolid was filtered, washed with water, and dried under vacuum toconstant weight. Compound A Form A was obtained as white to light yellowcrystalline solid.

Data

The XRPD pattern for Compound A Form A is shown in FIG. 1 and peaks andtheir related intensities in the XRPD pattern are shown in Table 1below.

TABLE 1 Peaks in the XRPD Pattern for Compound A Form A Peak Position(°2θ) Relative Intensity (%) 8.5 100 10.1 3.5 11.4 9.2 12.8 20.6 14.53.2 15.9 13.4 16.2 45.5 16.9 18.5 17.1 11.5 17.5 19.0 19.0 12.5 19.9 7.720.2 2.8 21.6 31.9 21.8 16.0 22.0 11.9 22.2 17.2 22.6 17.4 22.9 36.423.6 4.7 23.8 6.5 24.1 3.4 24.7 11.0 25.2 3.0 25.6 9.8 25.8 16.5 27.460.6 28.2 7.7 28.4 3.7 29.1 7.6 29.2 5.8 29.6 5.3 30.0 2.7 30.4 2.3 31.32.8 31.9 5.9 32.0 6.1 32.8 3.0 33.4 15.6 33.6 16.1 34.1 5.2 34.6 2.835.1 4.3 35.2 4.2 35.3 3.2 35.7 4.0 36.5 2.2 36.6 2.2 36.9 2.4 37.0 2.437.3 4.0 37.4 2.8 37.7 2.3 37.8 2.3 38.2 2.9 38.5 3.2 38.9 2.4 39.3 2.440.8 2.8 41.5 4.9

The results of the differential scanning calorimetry andthermogravimetric analyses of Compound A Form A are presented in FIGS. 2and 24, respectively. The thermogravimetric analysis shows negligibleweight loss of approximately 0.4% between 25° C. and 225° C., followedby a steady loss of weight above 225° C., suggesting sublimation ordecomposition of the material at these temperatures (FIG. 24). Thedifferential scanning calorimetry analysis of Compound A Form A showed avery shallow exotherm in the range from about 80-190° C., followed by asharp endotherm at about 224.3° C. (peak maximum). The sharp endothermcorresponded to the melt of the material, as determined by hotstagemicroscopy.

The hotstage microscopy of Compound A Form A showed little change of thematerial below its melting point. Some changes in birefringence werenoted in the range of about 150-200° C. The sample melted within thetemperature range of about 218.5-222.4° C.

The moisture sorption data for Compound A Form A showed negligibleweight gain, approximately 0.2% gained from between 5% to 95% relativehumidity, which was lost on desorption. The small moisture uptake ofCompound A Form A is indicative of a kinetically non-hygroscopicmaterial.

Example 2 Preparation of Compound A Form B Method

Crystalline Compound A Form B was provided by lyophilization of Form Ain a 1,4-dioxane:water (2:1) mixture. 20 mg of the crystalline CompoundA Form B was dissolved in 20 volumes 1,4-dioxane before addition of 20volumes cosolvent. The solvent systems were left to evaporate at roomtemperature, under the fume hood. The solids obtained from thisexperiment were analysed by XRPD.

Each of the following cosolvents used in the above described methodprovided Form B. 1,4-Dioxane:water (1:1), 1,4-dioxane:water (1:1),1,4-dioxane:methanol (1:1), 1,4-dioxane:ethanol, 1,4-dioxane:acetone(1:1), 1,4-dioxane:THF (1:1), and 1,4-dioxane:heptane (1:1).

Data

The XRPD pattern for Compound A Form B is shown in FIG. 3 and peaks andtheir related intensities in the XRPD pattern are shown in Table 2below. The crystalline pattern changed after the sample was stored at25° C./96% RH for twelve days, reverting to Form A.

TABLE 2 Peaks in the XRPD Pattern for Compound A Form B Peak Position(°2θ) Relative Intensity (%) 4.2 53.9 8.3 100 9.8 1.3 10.5 1.1 11.5 1.012.5 12.9 12.7 8.3 12.9 1.3 14.1 13.7 15.8 3.9 16.6 76.3 17.4 10.4 19.23.7 20.9 8.2 21.0 3.7 21.8 3.7 22.7 3.0 22.9 4.6 24.9 2.7 25.0 4.9 25.91.5 27.5 1.5 28.4 2.1 28.8 1.7 29.3 9.0 30.0 1.3 30.8 1.4 31.6 1.2 33.56.9 33.6 9.9 35.2 1.5 37.0 1.2 37.9 4.3 41.6 0.8

No residual solvent was observed, other than water. A weight loss of2.8% w/w between room temperature and 90° C. in the TGA thermogramsuggested the presence of 0.5 equivalent of water (theoretical 2.5% w/w)(FIG. 4). The DSC thermogram showed an endothermic event associated tothe weight loss (FIG. 4). A sharp endothermic event occurs at 222.3° C.(−127.8 J/g), which matches the melt of Form A.

High resolution XRPD data was collected over a month. After a full monthat ambient temperature (32 days), the sample (Form B) almost completelyreverted to anhydrous Form A.

Example 3 Preparation of Compound A Form C Method

Crystalline[(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid hexafluoropropan-2-ol solvate (Compound A Form C) was preparedfollowing the procedure described in Methods I, II, III and VI ofExample 1 using hexafluoropropan-2-ol as the solvent.

Data

The XRPD pattern for Compound A Form C is shown in FIG. 5 and peaks andtheir related intensities in the XRPD pattern are shown in Table 3below.

TABLE 3 Peaks in the XRPD Pattern for Compound A Form C Peak Position(°2θ) Relative Intensity (%) 4.5 100 8.5 1.6 9.1 7.9 10.2 1.9 11.2 2.511.8 1.2 12.5 1.2 13.7 37 15.4 9.2 15.5 8.3 15.6 3 16.4 11.5 16.9 2.517.7 3.3 18.3 2.7 18.7 1.2 19.0 2.3 19.8 2.5 20.6 8 21.9 3.5 22.4 1.522.5 2.3 22.9 6.3 23.3 3.1 23.8 3.4 24.6 2 25.2 2.3 25.4 3.1 25.7 1.926.0 1.5 27.5 1.2 27.9 2.3 28.4 2.6 29.1 1.4 29.4 1 29.8 1 30.2 1 30.82.1 31.6 1.2 31.7 1.5 32.2 1.3 32.3 1.1 33.1 0.9 34.1 1.4 34.2 1.5 35.10.9 35.5 0.8 37.0 0.9 37.8 1.1 38.7 1 40.2 0.7 40.9 1.1 41.8 1.2

Residual solvent was observed by proton NMR and was assigned to thehexafluoropropan-2-ol. Thermal analysis was also carried out on thissample (FIG. 6). A weight loss of 7.8% w/w between room temperature and130° C. in the TGA thermogram suggested the presence of 1/6 equivalentof hexafluoropropan-2-ol (theoretical 7.36% w/w) in the sample. The DSCthermogram showed an endothermic event associated to the weight lossfollowed by a small exothermic event (ca. 130° C.) (FIG. 6). A sharpendothermic event occurs at 222.2° C. (−17.9 J/g), which matches themelt of Form A. In conclusion, the material isolated fromhexafluoropropan-2-ol is a meta-stable solvate under ambient conditionsand converts to Form A.

Example 4 Preparation of Compound A Form D Method

Crystalline[(4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid DMSO:water solvate (Compound A Form D) was prepared from slowevaporation from THF/DMSO (20 volumes THF/5 volumes DMSO) of eithermostly amorphous Compound A or Form A.

Data

The XRPD pattern for Compound A Form D is shown in FIG. 8 and peaks andtheir related intensities in the XRPD pattern are shown in Table 4below. The sample (Form D) converted to Form A when left drying atambient temperature.

TABLE 4 Peaks in the XRPD Pattern for Compound A Form D Peak Position(°2θ) Relative Intensity (%) 4.2 32.7 4.3 29.4 8.4 65 8.5 65 12.6 30.915.8 20.6 16.8 100 18.9 5.5 19.3 19.1 20.7 6.2 21.7 3.8 22.1 23 22.6 4.623.1 11.7 24.0 8.7 24.5 3.1 25.3 12.1 25.4 10.3 26.8 4 27.1 4.4 27.3 3.928.4 43 31.2 7.7 32.0 3.4 33.3 4.4 33.4 3 34.0 6.2 35.8 3.7 38.3 7.639.1 3.6 40.9 2.5 41.6 3.6

Residual solvent was observed by proton NMR and was assigned to theDMSO. The TGA thermogram and DSC thermogram are shown in FIG. 8. The TGAshows a first weight loss between 40-150° C. of 18.5% (combination ofwater and DMSO) and a second weight loss between 170-220° C. (possibleDMSO). Two broad endotherms at 37.6° C. and 90.4° C. were possible dueto loss of water and DMSO. A small endotherm at 222.0° C. was observeddue to melt of Form A.

Example 5 Preparation of Salts of Compound A Method

The crystalline Compound A Form A was used to prepare the followingsalts. Compound A Form A (50 mg per experiment) was dissolved in acetoneor THF (50 vol, 2.1 ml) at 50° C. The solutions were treated with 1.1mol eq. of the corresponding counter ion (for example, 1.0 M aqueoussolution of sodium hydroxide, potassium hydroxide or hydrochloric acid).The temperature was maintained at 50° C. for 20 min then cooled to 0° C.at 0.1° C./min with stirring. After 20 h at 0° C., the solids werefiltered, air dried for 10 min and analyzed by the appropriatetechniques.

Data Compound A Sodium Salt

The XRPD pattern for Compound A sodium salt is shown in FIG. 9 and peaksand their related intensities in the XRPD pattern are shown in Table 5below.

TABLE 5 Peaks in the XRPD Pattern for Compound A Sodium Salt PeakPosition (°2θ) Relative Intensity (%) 5.3 22.0 8.1 4.4 11.1 10.9 13.59.3 15.1 5.2 16.0 100.0 17.0 9.6 18.7 20.0 19.2 13.3 21.6 30.7 22.9 7.724.0 12.6 25.3 7.5 26.2 11.0 28.9 10.1

The stoichiometry (ionic Compound A: counter ion) was determined to be1:1 by Ion Chromatography (Metrohm 761 Compact IC, IC Net softwarev2.3). The TGA thermogram and DSC thermogram are shown in FIG. 10. TheTGA thermogram shows a weight loss between 40-90° C. of 11.5%. The DSCthermogram shows a broad endotherm at 64.1° C., followed by twoexotherms at 150.5° C. and 190.3° C. and a sharp melt at 313.6° C.Purity was determined to be about 99.6%.

Compound A Potassium Salt

The stoichiometry (ionic Compound A: counter ion) was determined to be1:1 by Ion Chromatography (Metrohm 761 Compact IC, IC Net softwarev2.3). The XRPD pattern for the Compound A potassium salt is shown inFIG. 21. As seen in the Figure, the potassium salt is substantiallyamorphous. Thermal analysis showed a possible loss of water followed bya recrystallization event to produce non-solvated, crystalline form witha melt at 291° C. (FIG. 22).

Compound A L-Arginine Salt

The XRPD pattern for Compound A L-arginine salt is shown in FIG. 11 andpeaks and their related intensities in the XRPD pattern are shown inTable 6 below.

TABLE 6 Peaks in the XRPD Pattern for Compound A L-arginine salt PeakPosition (°2θ) Relative Intensity (%) 10.8 24.5 11.4 13.1 12.0 15.1 15.831.7 17.1 18.2 18.0 20.9 19.4 41.9 20.8 100.0 21.8 59.7 22.7 53.7 23.456.8 24.4 37.5 25.4 64.4 26.4 53.7 26.8 48.8 27.5 52.3 28.5 40.4 29.730.5

The stoichiometry (ionic Compound A: counter ion) was determined to beabout 1:1 by NMR. The TGA thermogram and DSC thermogram are shown inFIG. 12. The TGA thermogram shows a weight loss between 40-100° C. of4.5%. The DSC thermogram shows two broad endotherms at 79.6° C. and143.3° C., an exotherm at 172.5° C., followed by an endotherm at 210.1°C. Purity was determined to be about 99.5%.

Compound A L-Lysine Salt

The XRPD pattern for Compound A L-lysine salt is shown in FIG. 13 andpeaks and their related intensities in the XRPD pattern are shown inTable 7 below.

TABLE 7 Peaks in the XRPD Pattern for Compound A L-lysine salt PeakPosition (°2θ) Relative Intensity (%) 6.8 11.5 9.5 36.4 9.9 57.4 10.271.5 13.1 13.9 13.8 18.4 14.4 44.8 14.6 41.6 16.9 78.8 17.3 55.4 18.465.1 19.0 50.1 19.8 92.4 20.7 100.0 21.2 95.1 22.1 53.7 23.6 58.0 25.559.8 25.0 64.7 26.1 63.9 27.4 54.3 28.4 53.4 28.6 53.8 28.8 52.3 30.041.7 30.6 38.2

The stoichiometry (ionic Compound A: counter ion) was determined to beabout 1:1 by NMR. The TGA thermogram and DSC thermogram are shown inFIG. 14. The TGA thermogram shows a weight loss between 235-270° C. of12.1%. The DSC thermogram shows a sharp melt at 230.7° C. and a broadendotherm at 237.1° C. Purity was determined to be about 99.6%.

Compound A Ethanolamine Salt

The XRPD patterns for Compound A ethanolamine salt is shown in FIG. 15and peaks and their related intensities in the XRPD patterns are shownin Tables 8, 9 and 10 below. Pattern 1 was observed from acetone,pattern 2 was observed from THF, and Pattern 3 was observed at 40°C./75% RH.

TABLE 8 Peaks in the XRPD Pattern for Compound A ethanolamine salt(Pattern 1) Peak Position (°2θ) Relative Intensity (%) 3.8 15.6 4.6 6.35.1 8.3 7.7 4.8 10.9 32.8 12.5 10.0 15.0 28.2 15.5 23.5 17.9 14.0 18.620.1 21.1 45.2 21.8 54.9 22.7 77.7 24.4 30.3 26.2 53.6 26.6 47.6 27.1100.0

TABLE 9 Peaks in the XRPD Pattern for Compound A ethanolamine salt(Pattern 2) Peak Position (°2θ) Relative Intensity (%) 12.5 25.5 13.122.4 14.9 12.8 15.9 37.4 16.7 29.3 17.1 68 0 17.8 19.4 18.5 79.6 20.242.4 21.4 73.3 22.8 100.0 23.8 80.2 24.7 34.6 25.7 57.9 26.8 28.9 27.419.0 28.0 32.7 29.5 41.4 30.8 19.6

TABLE 10 Peaks in the XRPD Pattern for Compound A ethanolamine salt(Pattern 3) Peak Position (°2θ) Relative Intensity (%) 7.2 18.1 10.315.2 10.8 39.7 13.4 8.1 14.1 37.3 16.2 52.9 16.9 37.5 18.1 17.7 21.3100.0 21.7 64.8 22.3 21.7 22.9 42.3 23.1 39.6 23.7 36.1 25.3 29.9 26.223.3 26.9 50.5 27.8 75.0 29.0 22.7 18.5 11.8

For both THF and acetone, the stoichiometry (ionic Compound A: counterion) was determined to be about 1:1 by NMR. The TGA thermogram and DSCthermogram for the Compound A ethanolamine salt from acetone is shown inFIG. 16. The TGA thermogram shows a weight loss between 155-250° C. of10.1% (0.8 equivalents of ethanolamine). The DSC thermogram shows asharp melt at 171.4° C. and a broad endotherm at 186.0° C. Purity wasdetermined to be about 99.0%. For the Compound A ethanolamine salt fromTHF, the TGA thermogram showed a weight loss between 155-250° C. of10.1% (0.8 equivalents of ethanolamine), and the DSC thermogram showed asharp melt at 172.4° C. and a broad endotherm at 185.5° C. Purity wasdetermined to be about 99.1%.

Compound A Diethanolamine Salt

The XRPD patterns for Compound A diethanolamine salt is shown in FIG. 17and peaks and their related intensities in the XRPD patterns are shownin Tables 11 and 12 below. Pattern 1 was observed from acetone andPattern 2 was observed at 40° C./75% RH.

TABLE 11 Peaks in the XRPD Pattern for Compound A diethanolamine salt(Pattern 1) Peak Position (°2θ) Relative Intensity (%) 6.6 6.6 11.2 12.511.8 21.6 13.0 9.5 14.5 13.6 15.6 22.9 16.9 100.0 19.6 37.5 20.5 27.721.4 23.1 22.6 37.3 23.7 42.9 25.0 46.9 26.0 36.5 27.1 35.3 28.3 20.829.4 17.1 30.6 13.2

TABLE 12 Peaks in the XRPD Pattern for Compound A diethanolamine salt(Pattern 2) Peak Position (°2θ) Relative Intensity (%) 5.9 9.4 8.9 5.911.1 23.9 11.5 13.9 11.9 12.7 13.9 7.0 14.9 27.7 16.0 100.0 18.3 20.020.0 11.9 20.9 32.5 21.5 20.5 22.3 26.4 23.3 54.6 24.0 17.1 24.8 33.525.6 22.4 27.6 27.7 29.0 20.1

The stoichiometry (ionic Compound A: counter ion) was determined to beabout 1:1 by NMR. The TGA thermogram and DSC thermogram are shown inFIG. 18. The TGA thermogram shows a weight loss between 155-250° C. of12.6%. The DSC thermogram shows a sharp melt at 150.2° C. and a broadendotherm at 172.2° C. Purity was determined to be about 99.7%.

Compound A Tromethamine Salt

The XRPD pattern for Compound A tromethamine salt is shown in FIG. 19and peaks and their related intensities in the XRPD pattern is shown inTable 13 below.

TABLE 13 Peaks in the XRPD Pattern for Compound A tromethamine salt PeakPosition (°2θ) Relative Intensity (%) 9.4 4.3 10.1 100.0 11.8 8.5 13.415.3 14.2 53.0 15.0 5.1 16.9 20.3 19.2 12.9 20.1 23.5 21.1 69.2 22.321.4 23.4 14.7 24.3 13.6 25.1 10.5 25.7 21.5 26.3 16.9 28.4 22.9 29.212.0 30.0 17.1

The stoichiometry (ionic Compound A: counter ion) was determined to beabout 1:1 by NMR. The TGA thermogram and DSC thermogram are shown inFIG. 20. The TGA thermogram shows a weight loss between 180-260° C. of11.0%. The DSC thermogram shows a sharp melt at 176.5° C. and a broadendotherm at 182.6° C. Purity was determined to be about 99.7%.

Compound A Hydrochloric Acid Salt

The XRPD pattern for Compound A hydrochloric acid salt is shown in FIG.25 and peaks and their related intensities in the XRPD pattern is shownin Table 14 below.

TABLE 14 Peaks in the XRPD Pattern for Compound A hydrochloric acid saltPeak Position (°2θ) Relative Intensity (%) 8.2 6.4 10.2 18.3 10.7 13.612.5 10.9 13.6 36.1 19.0 27.1 19.8 21.1 20.3 30.6 20.9 42.7 21.3 17.422.5 68.5 24.1 100.0 27.6 36.4 29.0 17.9 25.6 21.5

The stoichiometry (ionic Compound A: counter ion) was determined to beabout 1:1 by Ion Chromatography. The TGA thermogram and DSC thermogramare shown in FIG. 26. The TGA thermogram shows a weight loss between100-170° C. of 6.5% and a second weight loss between 185-210° C. of3.4%. The DSC thermogram shows two small endotherms at 154.3 and 201.6°C., and a sharp melt at 223.0° C. Purity was determined to be about99.1%.

Compound A Sulfuric Acid Salt

The XRPD pattern for Compound A sulfuric acid salt is shown in FIG. 27to be a mixture of the salt and Form A. The peaks and their relatedintensities in the XRPD pattern is shown in Table 15 below.

TABLE 15 Peaks in the XRPD Pattern for Compound A sulfuric acid saltPeak Position (°2θ) Relative Intensity (%) 6.1 48.8 8.5 36.2 15.4 15.016.1 82.4 17.1 32.4 17.4 75.9 19.8 82.9 22.9 100.0 23.7 33.4 24.7 99.526.1 73.0 27.3 70.9 28.1 29.8 28.8 35.1 29.6 35.1

The TGA thermogram and DSC thermogram are shown in FIG. 28. The TGAthermogram shows a weight loss between 10-110° C. of 6.5% and a secondweight loss between 180-280° C. of 27.4%. The DSC thermogram shows threesmall endotherms at 31.8, 55.7 and 91.0 associated with the first weightloss, and a large, broad endotherm at 201.4° C. due to decomposition.

Compound A Methanesulfonic Acid Salt

The XRPD patterns for Compound A methanesulfonic acid salt is shown inFIG. 29 and the peaks and their related intensities in the XRPD patternsare shown in Tables 16 and 17 below. Pattern 1 was observed fromacetone, and pattern 2 was observed from THF. Pattern 1 reverted to FormA at 40° C./75% RH and pattern 2 reverted to a mixture of Form A andPattern 1 at 40° C./75% RH.

TABLE 16 Peaks in the XRPD Pattern for Compound A methanesulfonic acidsalt (Pattern 1) Peak Position (°2θ) Relative Intensity (%) 4.9 100.09.8 21.0 12.9 24.9 14.9 25.8 17.0 43.9 19.4 49.7 21.5 25.3 23.7 23.424.8 21.5

TABLE 17 Peaks in the XRPD Pattern for Compound A methanesulfonic acidsalt (Pattern 2) Peak Position (°2θ) Relative Intensity (%) 4.6 25.8 6.918.4 7.1 12.7 9.7 8.3 12.2 26.6 11.6 8.8 13.6 15.5 14.0 20.1 17.7 31.718.7 100.0 20.5 93.8 22.1 44.8 23.1 24.7 23.7 30.7 24.6 54.3 25.6 30.326.4 21.6 27.0 36.7 28.4 17.2 29.3 25.4 30.5 14.1

For both THF and acetone, the stoichiometry (ionic Compound A: counterion) was determined to be about 1:1 by NMR. The TGA thermogram and DSCthermogram for the Compound A methanesulfonic acid salt from THF isshown in FIG. 30. The TGA thermogram shows a weight loss between 40-100°C. of 2.1%. The DSC thermogram shows a small endotherm at 153.5° C. anda sharp endotherm at 166.9° C. Purity was determined to be about 99.3%.For the Compound A methanesulfonic acid salt from acetone, the TGAthermogram showed no weight loss until sample degradation at about 180°C., and the DSC thermogram showed an endotherm at 144.5° C. due tosample melt. Purity was determined to be about 98.9%.

Example 6 Preparation of Amorphous Compound A

Amorphous Compound A was obtained as follows. Crystalline Compound AForm A (500 mg) was dissolved in THF (1.5 mL) at room temperature. Thesolution was filtered to remove any residual crystalline material. Thesolvent was removed by fast evaporation in the rotary evaporator. Analiquot of the obtained solid was examined by XRPD. Alternatively,amorphous Compound A was obtained by lyophilization of Compound A Form Afrom a mixture of 1,4-dioxane:water (2:1 v/v) or by packing and sealingCompound A into a capillary tube, melting the sample on a Koflerhotbench at about 240° C. for one minute and cooling at ambienttemperature. An XRPD of amorphous Compound A is shown in FIG. 23.

Example 7 Preparation of Bis TEA Salt of Compound A Method

The crystalline Compound A Form A (50 mg) was dissolved in acetone (50vol) at 50° C. The solution was treated with 2.1 mol eq. oftriethylamine (TEA). The temperature was maintained at 50° C. for 20 minthen cooled to 0° C. at 0.1° C./min with stirring. After 72 at 0° C.,the solids were filtered, air dried for 5 min and analysed by XRPD. Thesolutions were set for slow evaporation at ambient conditions.

Data

The XRPD pattern for Compound A bis TEA salt is shown in FIG. 31 andpeaks and their related intensities in the XRPD pattern are shown inTable 18 below.

TABLE 18 Peaks in the XRPD Pattern for Compound A bis triethylamine saltPeak Position (°2θ) Relative Intensity (%) 5.5 4.2 9.2 5.0 10.1 29.411.7 13.2 13.3 8.0 13.7 17.3 14.5 42.5 14.7 52.1 15.2 9.3 15.7 14.3 16.316.0 17.0 36.9 18.4 11.9 19.4 31.5 20.4 21.7 20.7 27.3 22.6 100.0 22.923.9 23.9 29.9 24.8 14.6 25.5 38.5 26.3 16.7 27.2 37.1 28.1 62.2

The TGA thermogram and DSC thermogram are shown in FIG. 32. The TEA wasisolated as a bis salt, however the salt started to dissociate to Form Aafter one week storage at 40° C./75% RH.

Example 8 Preparation of Hemi Calcium and Magnesium Salts Method

The calcium and magnesium salts of Compound A were prepared by ionexchange from the sodium salt. Compound A (450 mg) was dissolved inacetone at 50° C. (50 vol, 22.5 ml). The solution was treated with 1.1mol eq. of sodium hydroxide (1 M solution in water). A suspension wasformed on addition then it was cooled to 0° C. at 0.1° C./min. After 48h at 0° C., the solid was filtered, air dried for 10 min and analysed byXRPD. The sodium salt (50 mg) was dissolved in MeOH (20 vol, 1 ml) atroom temperature. The solution was heated to 50° C. and then treatedwith the corresponding counter ion (1 M solutions in MeOH). The mixtureswere cooled to 0° C. at 0.1° C./min. After 24 h at 0° C., the solidswere filtered, air dried for 5 min and analyzed by XRPD (first crop).The liquors were kept and set for slow evaporation at ambient conditionsto provide a second crop. The materials isolated from the Ca⁺² and Mg⁺²hemi salt experiments crystallized after one week incubation at 40°C./75% RH.

Data

The XRPD patterns for the Compound A hemi calcium and magnesium saltsare shown in FIG. 33 and FIG. 35, respectively. The peaks and theirrelated intensities in the XRPD patterns are shown in Tables 19 and 20below.

TABLE 19 Peaks in the XRPD Pattern for Compound A hemi calcium salt PeakPosition (°2θ) Relative Intensity (%) 5.7 8.8 6.6 10.5 8.6 10.2 9.7 8.110.9 24.0 11.5 63.5 12.9 36.4 13.4 20.5 14.4 32.8 14.6 34.0 15.3 20.817.0 27.6 17.7 19.6 20.0 29.0 21.6 31.0 23.1 51.9 23.8 39.2 24.4 52.425.9 100.0 27.0 88.1 28.1 40.3 29.3 25.7

TABLE 20 Peaks in the XRPD Pattern for Compound A hemi magnesium saltPeak Position (°2θ) Relative Intensity (%) 5.0 12.7 7.1 37.2 7.5 24.98.0 30.0 10.0 30.2 11.4 35.4 11.9 37.5 12.6 33.9 ′12.9 37.5 15.9 45.417.8 50.4 18.9 44.4 20.1 67.6 21.0 59.0 21.7 67.9 22.8 100.0

The DSC thermograms for the Compound A hemi calcium and magnesium saltsare shown in FIG. 34 and FIG. 36, respectively.

Example 9 Single Crystal of Compound A Form A

Single crystals were grown from acetone with sufficient quality forstructure determination by single crystal X-ray diffraction.

The structure solution was obtained by direct methods, full-matrixleast-squares refinement on F² with weighting w⁻¹=σ2(F_(o)²)+(0.0697P)²+(0.3149P), where P=(F_(o) ²+2F_(c) ²)/3, anisotropicdisplacement parameters, empirical absorption correction using sphericalharmonics, implemented in SCALE3 ABSPACK scaling algorithm. FinalwR²={Σ[w(F_(o) ²−2F_(c) ²)²]/Σ[w(F_(o) ²)²]^(1/2)}=0.1376 for all data,conventional R₁=0.0467 on F values of 2496 reflections withF_(o)>4σ(F_(o)), S=1.045 for all data and 248 parameters. Final Δ/σ(max)0.000, Δ/σ(mean), 0.000. Final difference map between +0.211 and −0.318e Å⁻³.

FIG. 37 shows a view of a molecule of Compound A Form A from the crystalstructure showing the numbering scheme employed. Anisotropic atomicdisplacement ellipsoids for the non-hydrogen atoms are shown at the 50%probability level. Hydrogen atoms are displayed with an arbitrarilysmall radius.

TABLE 21 Samples Submitted for Single Crystal X-Ray Diffraction StudiesMolecular Formula C₁₉H₁₆N₂O₅ Molecular Weight  352.34 Crystal systemTriclinic Space group P-1 a  8.5208(13) Å, α 98.415(11)°, b  9.2233(13)Å, β 108.788(12)°, c 11.1859(14) Å γ 102.841(12)° V 788.50(19) Å³ Z   2D_(c) 1.484 g · cm⁻¹ μ 0.909 mm⁻¹ Source, λ Cu—Kα, 1.54178 Å F(000)  368T 100(2) K crystal Colorless prism, 0.11 × 0.05 × 0.02 mm data truncatedto 0.80 Å θ_(max) 77.18° Completeness 98.2% Reflections 12441 Uniquereflections  3282 R_(int)   0.0406

Example 10 Preparation of Compound A a) 5-Phenoxyphthalide

A reactor was charged with DMF (68 Kg), and stirring was initiated. Thereactor was then charged with phenol (51 Kg), acetylacetone (8 Kg),5-bromophthalide (85 Kg), copper bromide (9 Kg), and potassium carbonate(77 Kg). The mixture was heated above 85° C. and maintained untilreaction completion and then cooled. Water was added. Solid was filteredand washed with water. Solid was dissolved in dichloromethane, andwashed with aqueous HCl and then with water. Solvent was removed underpressure and methanol was added. The mixture was stirred and filtered.Solid was washed with methanol and dried in an oven giving5-phenoxyphthalide (Yield: 72%, HPLC: 99.6%).

b) 2-Chloromethyl-4-phenoxybenzoic acid methyl ester

A reactor was charged with toluene (24 Kg), and stirring was initiated.The reactor was then charged with 5-phenoxyphthalide (56 Kg), thionylchloride (41 Kg), trimethyl borate (1 Kg), dichlorotriphenylphosphorane(2.5 Kg), and potassium carbonate (77 Kg). The mixture was heated toreflux until reaction completion and solvent was removed leaving2-chloromethyl-4-phenoxybenzoyl chloride. Methanol was charged and themixture was heated above 50° C. until reaction completion. Solvent wasremoved and replaced with DMF. This solution of the product methyl2-chloromethyl-4-phenoxybenzoic acid methyl ester in DMF was useddirectly in the next step (HPLC: 85%).

c) 4-Hydroxy-7-phenoxyisoquinoline-3-carboxylic acid methyl ester (1a)

A reactor was charged with a solution of 2-chloromethyl-4-phenoxybenzoicacid methyl ester (˜68 Kg) in DMF, and stirring was initiated. Thereactor was then charged with p-toluenesulfonylglycine methyl ester (66Kg), potassium carbonate (60 Kg), and sodium iodide (4 Kg). The mixturewas heated to at least 50° C. until reaction completion. The mixture wascooled. Sodium methoxide in methanol was charged and the mixture wasstirred until reaction completion. Acetic acid and water were added, andthe mixture was stirred, filtered and washed with water. Solid waspurified by acetone trituration and dried in an oven giving 1a (Yieldfrom step b): 58%; HPLC: 99.4%). ¹H NMR (200 MHz, DMSO-d6) δ 11.60 (s,1H), 8.74 (s, 1H), 8.32 (d, J=9.0 Hz, 1H), 7.60 (dd, J=2.3 & 9.0 Hz,1H), 7.49 (m, 3H), 7.24 (m, 3H), 3.96 (s, 3H); MS-(+)-ion M+1=296.09

d) Methyl1-((dimethylamino)methyl)-4-hydroxy-7-phenoxyisoquinoline-3-carboxylate(1b)

A flask was charged with 1a (29.5 g) and acetic acid (44.3 g±5%), andthen stirred. Bis-dimethylaminomethane (12.8 g±2%) was slowly added. Themixture was heated to 55±5° C. and maintained until reaction completion.The reaction product was evaluated by MS, HPLC and ¹H NMR. ¹H NMR (200MHz, DMSO-d6) δ 11.7 (s, 1H), 8.38 (d, J=9.0 Hz, 1H), 7.61 (dd, J=9.0,2.7 Hz, 1H), 7.49 (m, 3H), 7.21 (m, 3H), 5.34 (s, 2H), 3.97 (s, 3H),1.98 (s, 3H); MS-(+)-ion M+1=368.12.

e) Methyl1-((acetoxy)methyl)-4-hydroxy-7-phenoxyisoquinoline-3-carboxylate (1c)

The solution of 1b from a) above was cooled below 25° C., at which timeacetic anhydride (28.6 g±3.5%) was added to maintain temperature below50° C. The resulting mixture was heated to 100±5° C. until reactioncompletion.

The solution of 1c and 1d from above was cooled to less than 65±5° C.Water (250 mL) was slowly added. The mixture was then cooled to below20±5° C. and filtered. The wet cake was washed with water (3×50 mL) andadded to a new flask. Dichloromethane (90 mL) and water (30 mL) wereadded, and the resulting mixture was stirred. The dichloromethane layerwas separated and evaluated by HPLC.

The organic layer was added to a flask and cooled 5±5° C. Morpholine wasadded and the mixture was stirred until reaction completion. Solvent wasreplaced with acetone/methanol mixture. After cooling, compound 1cprecipitated and was filtered, washed and dried in an oven (Yield: 81%,HPLC: >99.7%). ¹H NMR (200 MHz, DMSO-d6) δ 11.6 (S, 1H), 8.31 (d, J=9.0Hz, 1H), 7.87 (d, J=2.3 Hz, 1H), 7.49 (m, 3H), 7.24 (m, 3H), 3.95 (s,3H), 3.68 (s, 2H), 2.08 (s, 6H); MS-(+)-ion M+1=357.17.

f) Methyl 4-hydroxy-1-methyl-7-phenoxyisoquinoline-3-carboxylate (1e)

A reactor was charged with 1c (16.0 g), Pd/C (2.08 g), anhydrous Na₂CO₃(2.56 g) and ethyl acetate (120 mL). The flask was vacuum-purged withnitrogen (3×) and vacuum-purged with hydrogen (3×). The flask was thenpressurized with hydrogen and stirred at about 60° C. until completionof reaction. The flask was cooled to 20-25° C., the pressure released toambient, the head space purged with nitrogen three times and mixture wasfiltered. The filtrate was concentrated. Methanol was added. The mixturewas stirred and then cooled. Product precipitated and was filtered anddried in an oven (Yield: 90%, HPLC: 99.7%).

g) [(4-Hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid (Compound A)

A pressure flask was charged with 1e (30.92 g), glycine (22.52 g),methanol (155 mL), sodium methoxide solution (64.81 g) and sealed (as analternative, sodium glycinate was used in place of glycine and sodiummethoxide). The reaction was heated to about 110° C. until reaction wascomplete. The mixture was cooled, filtered, washed with methanol, driedunder vacuum, dissolved in water and washed with ethyl acetate. Theethyl acetate was removed and to the resulting aqueous layer an aceticacid (18.0 g) solution was added. The suspension was stirred at roomtemperature, filtered, and the solid washed with water (3×30 mL), coldacetone (5-10° C., 2×20 mL), and dried under vacuum to obtain Compound A(Yield: 86.1%, HPLC: 99.8%).

Example 11 Biological Testing

The solid forms provided herein can be used for inhibiting HIFhydroxylase activity, thereby increasing the stability and/or activityof hypoxia inducible factor (HIF), and can be used to treat and preventHIF-associated conditions and disorders (see, e.g., U.S. Pat. No.7,323,475, U.S. Patent Application Publication No. 2007/0004627, U.S.Patent Application Publication No. 2006/0276477, and U.S. PatentApplication Publication No. 2007/0259960, incorporated by referenceherein).

The biological activity of the solid forms provided herein may beassessed using any conventionally known method. In particularembodiments, cells derived from animal tissues, preferably humantissues, capable of expressing erythropoietin when stimulated bycompounds of the invention are cultured for the in vitro production ofendogenous proteins. Cells contemplated for use in such methods include,but are not limited to, cells derived from hepatic, hematopoietic,renal, and neural tissues.

Cell culture techniques are generally available in the art and includeany method that maintains cell viability and facilitates expression ofendogenous proteins. Cells are typically cultured in a growth mediumoptimized for cell growth, viability, and protein production. Cells maybe in suspension or attached to a substrate, and medium may be suppliedin batch feed or continuous flow-through regimens. Compounds of theinvention are added to the culture medium at levels that stimulateerythropoietin production without compromising cell viability.Erythropoietin produced by the cells is secreted into the culturemedium. The medium is then collected and the erythropoietin is purifiedusing methods known to those of skill in the art. (See, e.g., Lai et al.(1987) U.S. Pat. No. 4,667,016; and Egrie (1985) U.S. Pat. No.4,558,006.)

Suitable assay methods are well known in the art. The following arepresented only as examples and are not intended to be limiting.

Cell-Based HIFα Stabilization Assay

Human cells (e.g., Hep3B cells from hepatocellular tissue) derived fromvarious tissues were separately seeded into 35 mm culture dishes, andgrown at 37° C., 20% O₂, 5% CO₂ in standard culture medium, e.g., DMEM(Dulbecco's modification of Eagle's medium), 10% FBS (fetal bovineserum). When cell layers reach confluence, the media was replaced withOPTI-MEM media (Invitrogen Life Technologies, Carlsbad Calif.), and celllayers were incubated for approximately 24 hours in 20% O₂, 5% CO₂ at37° C. Compound A or 0.013% DMSO (dimethyl sulfoxide) was then added toexisting medium and incubation was continued overnight.

Following incubation, the media was removed, centrifuged, and stored foranalysis (see Cell-based VEGF and EPO assays below). The cells werewashed two times in cold phosphate buffered saline (PBS) and then lysedin 1 mL of 10 mM Tris (pH 7.4), 1 mM EDTA, 150 mM NaCl, 0.5% IGEPAL(Sigma-Aldrich, St. Louis Mo.), and a protease inhibitor mix (RocheMolecular Biochemicals) for 15 minutes on ice. Cell lysates werecentrifuged at 3,000×g for 5 minutes at 4° C., and the cytosolicfractions (supernatant) were collected. The nuclei (pellet) wereresuspended and lysed in 100 μL of 20 mM HEPES (pH 7.2), 400 mM NaCl, 1mM EDTA, 1 mM dithiothreitol, and a protease mix (Roche MolecularBiochemicals), centrifuged at 13,000×g for 5 minutes at 4° C., and thenuclear protein fractions (supernatant) were collected.

The Nuclear protein fractions collected were analyzed for HIF-1α using aQUANTIKINE immunoassay (R&D Systems, Inc., Minneapolis Minn.) accordingto the manufacturer's instructions.

Cell-Based EPO Assay

Hep3B cells (human hepatocellular carcinoma cells from ATCC, cat#HB-8064) were plated at 25,000 cells per well 96 well plates. The nextday, the cells were washed once with DMEM (Cellgro, cat #10-013-CM)+0.5%fetal bovine serum (Cellgro, cat #35-010-CV) and incubated with variousconcentrations of compound or vehicle control (0.15% DMSO) in DMEM+0.5%fetal bovine serum for 72 hours. Cell free culture supernatants weregenerated by transfer to a conical bottom 96 well plate andcentrifugation for 5 minutes at 2000 rpm. The supernatant wasquantitated for EPO using a human EPO ELISA kit (R&D Systems, cat #DEP00).

The EPO values for the compounds reported herein (e.g., Table 22) arethe measured value for cells plus compound minus the value for thevehicle control for the same cell preparation. The EPO values for thevehicle control for the cell preparations varied from 0-12.5 mIU/mL.

HIF-PH Assay

Ketoglutaric acid α-[1-¹⁴C]-sodium salt, alpha-ketoglutaric acid sodiumsalt, and HPLC purified peptide were obtained from commercial sources,e.g., Perkin-Elmer (Wellesley Mass.), Sigma-Aldrich, and SynPep Corp.(Dublin Calif.), respectively. Peptides for use in the assay werefragments of HIFα as described above or as disclosed in InternationalPublication WO 2005/118836, incorporated by reference herein. Forexample, a HIF peptide for use in the HIF-PH assay was[methoxycoumarin]-DLDLEALAPYIPADDDFQL-amide. HIF-PH, e.g., HIF-PH2 (alsoknown as EGLN1 or PHD2), was expressed in, e.g., insect Hi5 cells, andpartially purified, e.g., through a SP ion exchange chromatographycolumn. Enzyme activity was determined by capturing ¹⁴CO₂ using an assaydescribed by Kivirikko and Myllyla (1982, Methods Enzymol. 82:245-304).Assay reactions contained 50 mM HEPES (pH 7.4), 100 μM α-ketoglutaricacid sodium salt, 0.30 μCi/mL α-ketoglutaric acid α-[1-¹⁴C]-sodium salt,40 μM FeSO₄, 1 mM ascorbate, 1541.8 units/mL Catalase, with or without50 μM peptide substrate and various concentrations of compound of theinvention. Reactions were initiated by addition of HIF-PH enzyme.

The peptide-dependent percent turnover was calculated by subtractingpercent turnover in the absence of peptide from percent turnover in thepresence of substrate peptide. Percent inhibition and IC₅₀ werecalculated using peptide-dependent percent turnover at given inhibitorconcentrations. Calculation of IC₅₀ values for each inhibitor wasconducted using GraFit software (Erithacus Software Ltd., Surrey UK).The results are summarized in Table 22.

Table 21 below was intended to demonstrate the pharmacological utilityof Compound A. By inhibiting HIF prolyl hydroxylase enzymes (for examplePHD2, also known as EGLN1), Compound A stabilizes HIFα, which thencombines with HIFβ to form an active transcription factor that increasesexpression of numerous genes involved in response to hypoxic andischemic conditions, including erythropoietin (EPO). Therefore CompoundA can be used for the prevention, pretreatment, or treatment ofconditions associated with HIF and or EPO including anemic, ischemic andhypoxic conditions.

TABLE 22 IC₅₀ Cell EPO* PHD2 (μM) (mIU/mL) Compound A Form A 2.1 182*Cell EPO measured at 30 μM compound in DMSO compared to DMSO onlycontrol

1-54. (canceled)
 55. A pharmaceutical composition comprising crystalline[4-hydroxy-1-methyl-7-phenoxy-isoquinoline-3-carbonyl)-amino]-aceticacid (Compound A Form A) characterized by an X-ray powder diffractogramcomprising the following peaks: 8.5, 16.2, and 27.4°2θ±0.2°2θ, and apharmaceutically acceptable excipient, wherein at least about 85% ofCompound A is in Form A. 56-57. (canceled)
 58. The pharmaceuticalcomposition of claim 55, wherein at least about 90% of Compound A is inForm A.
 59. The pharmaceutical composition of claim 55, wherein at leastabout 95% of Compound A is in Form A.
 60. The pharmaceutical compositionof claim 55, wherein at least about 99% of Compound A is in Form A. 61.The pharmaceutical composition of claim 55, wherein at least about 99.5%of Compound A is in Form A.
 62. The pharmaceutical composition of claim55, wherein at least about 99.9% of Compound A is in Form A.
 63. Thepharmaceutical composition of claim 55, wherein at least about 99.99% ofCompound A is in Form A.
 64. A method for treating, pretreating, ordelaying onset or progression of a condition mediated at least in partby hypoxia inducible factor (HIF), comprising administering to a patientin need thereof, a therapeutically effective amount of thepharmaceutical composition of claim
 55. 65. The method of claim 64,wherein the condition mediated at least in part by HIF is tissue damageassociated with ischemia or hypoxia.
 66. The method of claim 65, whereinthe ischemia is associated with an acute event selected from the groupconsisting of myocardial infarction, pulmonary embolism, intestinalinfarction, chronic kidney failure, ischemic stroke, and renalischemic-reperfusion injury.
 67. The method of claim 65, wherein theischemia is associated with a chronic event selected from the groupconsisting of cardiac cirrhosis, transient ischemic attack, maculardegeneration, peripheral artery disease, and congestive heart failure.68. A method for treating, pretreating, or delaying onset or progressionof a condition mediated at least in part by erythropoietin (EPO),comprising administering to a patient in need thereof, a therapeuticallyeffective amount of the pharmaceutical composition of claim
 55. 69. Amethod for treating, pretreating, or delaying onset or progression ofanemia, comprising administering to a patient in need thereof, atherapeutically effective amount of the pharmaceutical composition ofclaim
 55. 70-71. (canceled)
 72. The method of claim 64, 68 or 69,wherein at least about 90% of the Compound A in the pharmaceuticalcomposition is in Form A.
 73. The method of claim 72, wherein at leastabout 95% of the Compound A in the pharmaceutical composition is in FormA.
 74. The method of claim 72, wherein at least about 99% of theCompound A in the pharmaceutical composition is in Form A.
 75. Themethod of claim 72, wherein at least about 99.5% of the Compound A inthe pharmaceutical composition is in Form A.
 76. The method of claim 72,wherein at least about 99.9% of the Compound A in the pharmaceuticalcomposition is in Form A.
 77. The method of claim 72, wherein at leastabout 99.99% of the Compound A in the pharmaceutical composition is inForm A.
 78. The pharmaceutical composition of claim 55, wherein thediffractogram further comprises peaks at 12.8, 21.6, and 22.9°2θ±0.2°2θ.79. The pharmaceutical composition of claim 55, wherein thediffractogram is substantially as shown in FIG.
 1. 80. Thepharmaceutical composition of claim 55, wherein the Compound A Form A ischaracterized by a differential scanning calorimetry (DSC) curve thatcomprises an endotherm at about 223° C.
 81. The pharmaceuticalcomposition of claim 80, wherein the DSC curve is substantially as shownin FIG. 2.